Naturally Occurring TAP-Dependent Specific T-Cell Tolerance for a Variant of an Immunodominant Retroviral Cytotoxic T-Lymphocyte Epitope

Abstract

Upon immunization and restimulation with tumors induced by the endogenous AKR/Gross murine leukemia virus (MuLV), C57BL/6 mice generate vigorous H-2Kb -restricted cytotoxic T-lymphocyte (CTL) responses to a determinant (KSPWFTTL) derived from the p15E transmembrane portion of the viral envelope glycoprotein. By contrast, the highly homologous determinant RSPWFTTL, expressed by tumor cells induced by Friend/ Moloney/Rauscher (FMR) MuLV, is not immunogenic, even when presented to the immune system as vaccinia virus-encoded cytosolic or endoplasmic reticulum (ER)-targeted minigene products. Such minigene products are usually highly immunogenic since they bypass the need for cells to liberate the peptide or transport the peptide into the ER by the transporter associated with antigen processing (TAP). Using KSPWFTTL-specific CTLs that cross-react with RSPWFTTL, we previously demonstrated that presentation of RSPWFTTL from its natural viral gene product is TAP dependent. Here, we show first that C57BL/6 mice express mRNA encoding RSPWFTTL but not KSPWFTTL and second that the ER-targeted RSPWFTTL minigene product is highly immunogenic in C57BL/6 mice with a targeted deletion in TAP1. These findings provide the initial demonstration of TAP-dependent tolerance induction to a specific self peptide and demonstrate that this contributes to the differential recognition of RSPWFTTL and KSPWFTTL by C57BL/6 mice.

Endogenously Expressed Antigens Bind Mammalian RNA via Cationic Domains that Enhance Priming of Effector CD8 T Cells by DNA Vaccination

“…Using well-defined primer pairs, we could not detect expression of AKV-gp70 mRNA in the thymus or spleen of BALB/c and B6 mice ( Figures 5 A and 5B). 29 , 31 , 37 However, retroviral gp70 mRNA was efficiently expressed in human and murine tumors and tumor cell lines, 38 for example, in the BALB/c-derived colon carcinoma CT26 29 and in B6-derived pancreatic ductal adenocarcinoma (AKC-5615 cells), established from a highly aggressive tumor in ATM-deficient AKC mice (Atm lox/lox ;Kras LSL-G12D/+ ;p48Cre; Atm lox/lox ( Figures 5 A and 5B). 39 To confirm that the expression of the RNA-binding tat domain in the stgp70tat antigen is sufficient to induce tumor-specific effector CD8 + T cell responses, we vaccinated B6 mice with pCI, pCI/stgp70, or pCI/stgp70tat DNA.…”

“…gp70-specific PCRs were performed with the primer sets gp70/AKV (5′-CGA GCC AAA TAT AAA AGA GAA CCC-3′/5′-GGT GGT GAA CCA AGG GGA CTT-3′) and β-actin (5′-CAT GTT TGA GAC CTT CAA CAC CC-3′/5′-GCC ATC TCC TGC AAG TCT AG-3′) and additionally (623/7499 5′-GTA CGG GAT AGC ATG GCC AAA CTT AGA GAA-3′; 623/7694 5′-CTA CCG AAA TCC TGT CTT TGA TAA ACT G-3′). 31 All primers were purchased from biomers.net (Ulm, Germany). Cycling parameters were as follows: (I) 1 cycle: 95°C, 10 min; (II) 30 cycles: 95°C, 10 s, 53°C, 30 s, 72°C, 1 min; and (III) 72°C, 10 min.…”

“…In this study, we tested de novo priming of antiviral K b /C93-specific effector CD8 + T cells in 1.4HBV-S mut tg mice by DNA vaccines expressing RNA-binding or RNA-free HBV core antigens. We further analyzed whether expression of K b /p15E or L d /AH1 epitopes, from a tumor-specific envelope gp70 antigen of an endogenous murine leukemia virus (AKV), 27 , 28 , 29 , 30 , 31 in RNA-binding model antigens affects priming of effector CD8 + T cells in mice by DNA vaccination.…”

Pseudomonas aeruginosa Cif Protein Enhances the Ubiquitination and Proteasomal Degradation of the Transporter Associated with Antigen Processing (TAP) and Reduces Major Histocompatibility Complex (MHC) Class I Antigen Presentation

“…Antigen Presentation Assays-Presentation of the SIINFEKL peptide was assessed using EL-4 cells treated in the presence or absence of P. aeruginosa (ϩCif) OMVs or (ϪCif) OMVs, then inoculated with PR8/SIINFEKL influenza at a multiplicity of infection of 10 and processed for flow cytometry by indirect labeling using the 25.D1-16 clone antibody to recognize specific K b -SIINFEKL complexes (12)(13)(14). Untreated cells were exposed in parallel with 1 nM OVA peptide as a positive control for 25.D1-16 clone antibody labeling.…”

IFN-γ treatment protocol for MHC-Ilo/PD-L1+ pancreatic tumor cells selectively restores their TAP-mediated presentation competence and CD8 T-cell priming potential

“… 23 30 38 In contrast to Cr-1:K b /cr 16-24 , the gp70:K b /p15E epitope is not located in the ER-targeting SP (SP gp70 ) ( figure 2A ) and its processing/presentation apparently depends on proteasomal processing 39 and TAP. 38 Therefore, this antigen/epitope needs retrotranslocation from the ER to the cytosol and/or ‘cross-presentation’ for efficient MHC-I processing/presentation. 20 40 Neither full-length gp70 nor epitope-containing gp70 fragments induced CD8 T cells in H-2 d BALB/c or H-2 b B6 mice.…”